This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our previously developed method of direct coupling of thin layer chromatography (TLC) with Vibrationally-Cooled (VC) MALDI-FTMS allows simple handling of TLC with FT high resolution which is not affected by the irregular surface of the TLC plate. Use of vibrational cooling is necessary for ganglioside stabilization and detection. Optimization of this technique has led to high sensitivity for the ions desorbed directly from the TLC plate. MALDI-FTMS experiments have been carried out on our home-built FT-ICR mass spectrometer with 7T magnet and Nd:YAG (UV) laser, frequency tripled to 355 nm, nitrogen laser (337 nm) and Er:YAG (IR) laser (2940 nm). The collision cooling gas raises the pressure in the ion source during the desorption-ionization event. Internal Calibration on Adjacent Samples (InCAS) is performed using standards desorbed from the stainless steel plate as calibrants. The sensitivity the has been obtained for the gangliosides desorbed from TLC is well below 1 pmole (total amount spotted on a TLC plate prior to separation). The use of InCAS has demonstrated that accuracy is not affected upon desorption of the analyte from uneven TLC plate surface and mass accuracy is usually 0.5-1.5 ppm for ganglioside standards. The effect of several parameters on molecule stabilization has been studied, such as the type of the cooling gas, gas pulsing coordinating with ionization-desorption event, cooling gas pressure, duration of gas pulse, different laser intensities, focusing and beam profile. Soft IR-desorption of the gangliosides with urea as the matrix, together with cooling in the Vibrationally Cooled source eliminated sialic acid loss. The developed method is being applied to the samples from biological systems such as whole brain ganglioside mix, Leishmania lipids, ganglioside-rich lipid rafts from neuronal cells and glycolipids from human milk. Infrared multiphoton dissociation (CO2 laser, 10.7 um) and SORI-CAD are being used as complementary dissociation methods. Because of source modifications, new TLC-MS data was not obtained during the current year, but progress on manuscripts was made and advice and guidance was provided to several groups that are undertaking or have plans for similar experiments.